Structural and Functional Investigation of a Type III CRISPR Immune Pathway and<br> a Bacterial Sialic Acid TRAP Transporter

Abstract

Multiple mechanisms have evolved to protect organisms from invaders. These immune strategies vary widely in complexity, specificity, response time, and long-term protection. While all types of immune systems continuously optimize and adapt for different applications, invaders also developed strategies to evade, or even actively counteract, the host immune response, ending up in a highly dynamic arms race of increasingly sophisticated attack and defense strategies. Beyond its evolutionary relevance, the significant conservation of innate immune mechanisms among prokaryotes and eukaryotes may help to unravel human defense strategies by characterizing their bacterial counterparts. On the other hand, studying bacterial strategies to evade host immune mechanisms can help to fight pathogens more specifically and effectively. This work contains two independent parts, part one focusses on tripartite ATP-independent periplasmic (TRAP) transporters, which help pathogenic bacteria like <em>Haemophilus influenzae</em> and <em>Vibrio cholerae</em> to survive in the host environment. Those bacteria rely on the import of sialic acid by TRAP transporters, that employ dedicated substrate binding proteins (SBPs) contributing to a selective and efficient transport process by binding the substrate and delivering it to the transporter. A better understanding of the TRAP transporter-mediated transport mechanism may help to combat these pathogens more efficiently. A structural and biophysical characterization of several VHH antibody/SBP complexes revealed an allosteric mechanism that does not only inhibit the high-affinity binding of sialic acid, but also triggers the release of already bound sialic acid from the binding pocket. The results revealed a previously unnoticed surface cavity as a key element in the conformational rearrangement of the SBP upon sialic acid binding, shedding new light on the structural mechanism of TRAP transporters, and providing evidence for a novel substrate release strategy. <br /> The second part characterizes a new CRISPR based antiviral signaling cascade. CRISPR systems represent one of the most prominent groups of prokaryotic immune systems and contain multiple subtypes that differ in their mode of action. Type III CRISPR systems enable a highly complex and versatile immune response via the synthesis of cyclic oligoadenylate (cOA) second messengers, which activate different effector proteins with diverse functions, ranging from RNases to transcriptional regulators to DNases. Bioinformatics studies have reported an unusual effector protein, a membrane-bound “CRISPR-associated Lon protease” (CalpL). The structural and mechanistic analysis of this ₄ activated protease and the associated phage defense system revealed that, unlike predicted, CalpL is a soluble monomeric protein that is activated by ₄ induced oligomerization. The activated protease cleaves the CalpT/S complex, two small proteins that are encoded in the same operon, directly upstream of CalpL. We found that ₄ activates CalpL to cleave the anti-sigma factor CalpT, releasing the sigma factor CalpS from the complex and allowing it to bind to RNA polymerase to regulate cellular transcription. This finding directly links a type III CRISPR system to the transcription machinery of the cell. Our results show similarities to the CBASS system, recent reports on CRISPR-activated caspases (Craspases), and even to mammalian systems such as the cGAS-STING pathway.